Method for preventing the adhesion of particles

ABSTRACT

A method for the prevention of the adhesion of particles, in particular cells and cellular components in solution to surfaces, characterized in that to the solution is added at least one polyalcohol.

The invention relates to a method for the prevention of the adhesion ofparticles, in particular of cells and cellular components, to surfaces,whereby a polyalcohol is added to the solution which contains theparticles.

The adherence to surfaces is a fundamental property of cells. Thisbecomes apparent, for example, in the interaction of cells with anextracellular matrix, which is an important factor in controlling thegene expression or general cell function. These processes are influencedin particular via proteins on the cell surface.

Indeed, this property of cells often presents a big problem during thepreparation of cell cultures and the nondestructive examination of cellsor cellular components. An adherence of cells to, for example, surfacesof the accessories and containers used in the preparation isundesirable. Technologies for the microanalysis of biological samples inparticular can not be utilized for cell examinations without problems.These systems that contain, for example, canal systems like in the“Lab-on-Chip”-technology, often only have dimensions of a fewmicrometers. Furthermore, the selection of the surface materials to beused of these microanalysis systems is bound to the particularmanufacturing process or the material of the microstructures in itself.Microchannel analysis systems, for example, are made from glass,silicon, silicone, and organic polymers, such as PMMA or polyurethane,all substrates that facilitate the adhesion of cells. The surfacematerial consequently can not be freely chosen according to therequirements for a minimal cell adhesion.

In order to prevent cell adherence/adhesion, the surface of themicrostructures in itself is therefore modified. Various chemicalcompounds, in general hydrophobic silanes and hydrophilic polymers(hydrogels) are used for this purpose.

Carlson et al. ((1997): “Self-Sorting of White Blood Cells in a Lattice”in “Physical Review Letters” 79, No. 11, p. 2149-2152) succeeded withthe known coatings in the sorting of blood cells, for example, inmechanical microfilter systems. But white blood cells therebyirreversibly adhered to the entrances of microchannels. This isdesirable in the described system, but at the same time shows thepresent limitations in the use of microchannels for the nondestructiveexamination of cellular samples.

Further investigations by Li et al. (“Transport, Manipulation, andReaction of Biological Cells On-Chip Using Electrokinetic Effects” in“Analytical Chemistry” (1997) 69, 1564-1568) showed that blood cells canbe brought into 40 μm-wide microchannels. But the cells weresubsequently lysed with a reagent and only then subjected to ananalysis. However, the recovery of eukaryotic, particularly of livingmammalian cells, from microchannels makes high demands on the selectionof the surface materials of the channels.

Further investigations, for example the coating of glass lamina withpolyvinyl alcohol, showed that such a surface modification can minimizean adhesion of mouse fibroblast (Hisada, T. (1976): “The adhesion ofculture cells to some polymers” (Japanese language) in “Shika RikogakuZasshi” 17(38), p. 91-101)).

It could likewise be shown, that copolymer particles from poly(methylmethacrylate) and polyvinyl alcohol do not adhere to blood cells and arenot phagocyted by monocytes and neutrophiles (Ayhan, H. and E. Piskin(1997): “Interaction of activated leukocytes with polymericmicrospheres” in “International Journal of Artificial Organs” 20(12),704-707).

Krylov et al. (in “Electrophoresis”, 21, 767-773, 2000) furthermorefound, that the adherence of cells to glass or polystyrene is markedlyreduced, if the surfaces are modified with hydrophilic polymers(hydrogels) such as polyvinyl alcohol.

However, in all the mentioned investigations, it is necessary to carryout a direct surface coating, whereby, for example, polyvinyl alcohol(PVA) is immobile bound to the surface, that is a surface coating or acopolymerization of a substrate with PVA is carried out. For theimplementation of the coating, complicated, in particular to exacttemperature specifications bound, multistep and thereby verytime-consuming processes are necessary. Furthermore, particularly thecoating of surfaces that consist of polymers such as polystyrene isproblematic, since this material can be damaged by the temperatures offar above 100° C. that are necessary for the coating. The method cantherefore only be satisfyingly applied to the use with glass surfaces.

Furthermore, it is often impossible, in particular due to the only smalldiameters of the microstructures in microanalysis systems, to achieve acoating that is satisfying. Thus, for example, a uniform distribution ofthe polymers or other coating materials across the surfaces of thecorresponding microstructures, for example of channel structures of adiameter of only a few micrometers, is not or only insufficientlypossible.

It is therefore the object of the present invention to develop a method,that independent of the nature and surface properties of a microanalysissystem allows an essentially nondestructive and loss-free examination ofparticles, particularly of cells and cellular components, in solution inthese systems.

The object is solved by a method according to claim 1 and a solutionaccording to claim 12.

The method comprises according to the invention the addition of asubstance to the solution that contains the particles, particularlycells or cellular components, but also, for example, may containproteins and peptides, that are present in the free form or bound tostructures such as, for example beads. This substance according to theinvention causes the adherence of these particles to surfaces,particularly surfaces of a microanalysis system, to be essentiallyprevented.

In the only figure, a diagram is shown that illustrates the recoveryrate of suspended Jurkat cells from a microliter-syringe versus theresidence time t in the presence of 0.5% PVA.

According to the invention this substance involves a polyalcohol, inparticular polyvinyl alcohol, for example polyvinyl alcohol VA30,000-70,000 and/or polyglycol, preferably polyethylene glycol.Surprisingly, the addition of this substance to the solution did notshow a toxic effect relating to living cells or does not cause change ofcellular components. Especially suitable is a concentration of thepolyalcohol in solution of 0.01% (w/v) to 5% (w/v). Consideringpolyvinyl alcohol as an example, it could even be shown, that theanti-adhesion effect of the polyalcohol by far exceeds that of knownsubstances that are used as anti-adhesives (table 3). The watersolubility and additional properties, listed below, of the polyalcoholcause a universal applicability in all aqueous particle solutions.

An additional important property of this substance, that in particularqualifies it for the use in solutions which contain cells and/orcellular components, is that it has no influence on the physiologicpH-value of the solution.

Many methods of analysis, that are applied in microanalysis systems, arebased upon the recording and evaluation of optical signals, such as, forexample, fluorescence correlation spectroscopy, fluorescence resonanceenergy transfer, fluorescence polarization, fluorescence intensitydistribution analysis, or, for example, UV-spectroscopy. Therefore, itwas all the more important for the universal applicability of thissubstance, that it does not influence an optical analysis, i.e.particularly does not show autofluorescence or change the refractiveindex of the solution. These requirements are very well met bypolyalcohols, particularly polyvinyl alcohols or polyglycols.

The method according to the invention now allows an essentiallyloss-free examination and manipulation, particularly separation, of theparticles, particularly in microanalysis systems.

Hereby it may concern microanalysis systems that have, for example,channels with a height of from 1-1,000 μm, particularly 2-500 μm,especially preferred 40 μm; a width of 100-2,000 μm, particularly200-600 μm, and a length of 100 μm-10 cm, preferably 1-2 cm. The systemsmay consist in particular of glass, silicon, silicone, organic polymers,or another suitable material. Specific embodiments of the microanalysissystems may also have electrodes, in particular for the generation ofdielectric forces for the manipulation of the particles that are to beexamined, for example for the microscopic, optical and/or electricalanalyzing, for the sorting, separating, electroporating, fusing andseparating of the particles, particularly of cells.

Microanalysis systems, for example, thus comprise electrodes asdielectric junctions in the microchannels for the sorting of suspendedparticles (in particular cells), or in other microanalysis systems theelectrodes are arranged in such a way that the cell fusion and/or cellporation can be carried out, and other systems comprise two electrodelevels in a closed microchannel, a dielectric field cage in a channelintersection as well as electrodes developed as dielectric junctions forthe implementation of a stop-flow analysis as well as a subsequentsorting of the analyzed particles. Whereby, the microchannel systems mayalso comprise combinations of the various electrode systems.

Even the injection of the particles into such a system, which is oftencarried out with the help of a plastic or glass syringe, does no longerlead to losses, which are caused by the adherence of the particles tothe syringe body.

Since the added substance according to the invention also does notinfluence a living cell, it is, for example, possible, to directlyexamine cells, to sort on the basis of the examinations and then tospecifically recreate cell cultures with the separated cells. That way,for example, cells with a certain property, for instance with a certainkind of receptors on the cell surface, are specifically and essentiallyloss-free separated from other cells and multiplied.

The method according to the invention is in general suitable for allcell types, consequently both for eukaryotic cells and for prokaryoticcells.

In table 1, the portion of living cells after the examination and/ormanipulation in various microanalysis systems in the presence ofpolyvinyl alcohol is shown. (Jurkat-cells in Cytocon™ sorter chips (No.1 and 2), in Cytocon™ porator chips (No. 3-5), and Cytocon™ loader chips(No. 6 and 7)).

TABLE 1 Flow Rate in μl/h Sheath Cell Amount Recovery No. Sample fluidPer μl Total Rate % 1 10 288 660 440 84 2 10 288 132 220 64 3 10 288 68974 4 10 144 144 100 5 10 144 144 113 6 18 144 160 160 87.3 7 20 18 305243 63

In table 2, in each case, the portion of Jurkat-cells and blood cells inthe fractions 1 and 2 after a cell separation in a microanalysis system(Cytocon™ chip) is shown.

TABLE 2 Number of Cells Total Fraction 1 Fraction 2 Total Jurkat RBCTotal Jurkat RBC Total Jurkat RBC 6,355 161 6,194 345 151 194 6,010 106000 100% 2.5% 97.5% 100% 43.8% 66.2% 100% 0.17% 99.8%

The method according to the invention is likewise suitable for cellularcomponents. It has no influence on their structure and does not causedegeneration. Under cellular components are to be understood, forexample, liposomes, lipid membranes, lipids, proteins, DNA, nucleicacids, messenger substances, sugar, glycans, and/or glycoproteins. Inprinciple, all components of a cell are thus concerned, but also cellfragments.

The method according to the invention provides, besides the advantagethat it prevents an adherence of the particles to the surfaces ofmicroanalysis systems, that may in particular consist of silicon,silicone, glass, and/or organic polymers such as PMMA, polyurethane,polycarbonate, polypropylene, polystyrene, polyethylene, polyvinylchloride, Teflon®, polyacryl (fiber), nylon®, and/or perlon®, also thepossibility that it prevents the adhesion of biomolecules, in particularcells, cell membranes, cellular components such as lipids, proteins (inparticular antibodies), DNA, nucleic acids, messenger substances,biotin, sugar, glycans, and/or glycoproteins among one another or tosurfaces coated with such molecules.

In table 3, the cell adherence to polystyrene culture dishes in thepresence of various substances is shown.

TABLE 3 Jurkat U937 Adherence Vitality Adherence Vitality RPMI —  97%  9100%   10% FBS PBS 999  99% 999  98% PBS + Ca, Mg 999 OK 999 OK  0.1%BSA  99 OK 999 OK  0.5% BSA — OK  99 OK   1% PVP 15  99 100% 999 100%  1% PVP 25  99 100% 999 100%   1% PEG 5,000  99 100% 999 100%   1% MC15  99 OK 999 OK   1% MC 400  99 OK  99 OK   10% Ficoll 70 999 OK 999 OK  10% Ficoll 400 999 OK 999 OK   10% 999 OK 999 OK Dextran T10   1% PVA—  97% (9)  98% 30,000–70,000  0.1% PVA — OK (9) OK 30,000–70,000 0.01%PVA — OK (9) OK 30,000–70,000 Notes: — =no adherence (9) = very lowadherence 9 = distinct adherence 99 = strong adherence 999 = very strongadherence OK = no quantification, but no difference to the controlnoticeable

Whereby within the meaning of this invention by surface is meant anysurface, but in particular channel surfaces, surfaces of storagecontainers of microanalysis systems, tubing, syringes, injectionmodules, or surfaces of synthetic microparticles.

Furthermore, a solution was found, that in general is suitable for theexamination of particles, in particular of cells and cellular componentsin microanalysis systems. This solution contains saline buffer, inparticular PBS, and/or inositol, and polyvinyl alcohol. However, to thesolution may also be added other components known in the context of cellsolutions such as, for example, magnesium and calcium ions.

The portions of saline buffer, inositol, and/or PVA are variable. Thebuffer may be diluted in a variable mixing ratio by normoosmolar,hyperosmolar, or hypoosmolar sugar solutions (for instance sucrose orinositol). The portion of saline buffer in the solution may be variedbetween 0 and almost 100%, whereby portions of 20%, 50%, or almost 100%are especially preferred.

Inositol is preferably used as a 0.2 to 0.5 M solution, for instance ofinositol in distilled water, especially preferred a 0.3 M solution.Whereby, the portion of inositol solution and the solution according tothe invention may also be varied between 0 and almost 100%. Especiallypreferred are indeed portions of 50%, 80%, and almost 100%.

Polyvinyl alcohol with a molecular weight of between 30,000 g/mol and70,000 g/mol is particularly well suited for the solution according tothe invention, whereby the amount of PVA in the solution according tothe invention lies between 0.1% (w/v) and 1.1% (w/v), preferably between0.3% (w/v) and 0.6% (w/v), especially preferred between 0.45% (w/v) and0.55% (w/v).

The solution according to the invention may be utilized independent oftype and properties of the particles, in particular for theirexamination and manipulation in microanalysis systems.

For this purpose, the particles may be directly suspended in thissolution and placed in the microanalysis system. However, it is alsopossible to add this solution to particle suspensions.

EXAMPLE 1

Influence of Polyvinyl Alcohol on the pH-Value of a Solution

The pH value of a 1% solution of polyvinyl alcohol in PBS was measuredand lies at 7.31 (the physiologic pH range is pH 7.2-7.4).

EXAMPLE 2

The Influence of Polyvinyl Alcohol on the Cell Adhesion in Comparison toOther Additives

Solutions:

Jurkat-cells (clone E6-1 from the European Collection of Animal CellCultures, Salisbury, England) and U937-cells (monocytic cell line of theEuropean Collection of Animal Cell Cultures, Salisbury, England) werecultivated in RPMI 1640 medium ((GIBCO Life Technologies, Karlsruhe) byadding 100 IU/ml each of penicillin and streptomycin (Seromed/Biochrom,Berlin) and 10% fetal calf serum (Seromed/Biochrom, Berlin). 3 ml eachof the cell suspension were centrifuged off and taken up in 300 μl ofphosphate-buffered saline solution without calcium or magnesium (PBS,Seromed/Biochrom, Berlin). The wells of a 24-well plate (24-wellpolystyrene culture plates, Corning Costar) were each filled with 500 μlPBS by adding the table 3 indicated concentrations of the subsequentlylisted substances. Methylcellulose (15 and 4,000 centipoise viscosity ofa 2% solution, SIGMA Aldrich GmbH, Steinheim), Ficoll 70 and Ficoll 400(Pharmacia, Uppsala, Sweden), Dextran T10 (Pharmacia, Uppsala, Sweden),polyvinyl pyrrolidone 15 and 25 (Serva), polyethylene glycol 5,000(Fluka) and polyvinyl alcohol 30,000-70,000 (SIGMA Aldrich GmbH,Steinheim) were tested. 50 μl of the cell suspension described abovewere added. A sample in 500 μl cell culture medium (in which adherenceof these suspension cells does not occur) served as control.

Implementation:

The samples were incubated for 30 minutes at 37° C. The supernatant withnon-adhered cells was removed and replaced by buffer. The adherence wasqualitatively evaluated (refer to table 3). The cells in bothsupernatant and the wells were stained with trypanblue solution (0.2%,SIGMA Aldrich GmbH, Steinheim) in order to examine the vitality of thecells. Only polyvinyl alcohol has a very distinct inhibitory effect onthe cell adherence with both cell lines. The effect already occurs at aconcentration of 0.1% (w/v).

Result:

Many substances, to which an anti-adhesive effect is assigned, weretested in comparison to polyvinyl alcohol. Bovine serum albumin (BSA),for instance, which is known from flow cytometry for the blocking ofnon-specific bonds and used in immunoreactive detection (e.g. Westernblot technique). Furthermore, various hydrophilic polymers that are usedin density gradient centrifugation of blood samples or in cell culturetechniques were examined. Even the influence of cadherins and integrins,specific cell-cell and cell-substrate adhesion molecules, in bufferswithout calcium and magnesium were examined. These molecules needcalcium for interaction. It became apparent, that polyvinyl alcoholdistinctly surpasses the other substances in the effect of successfulpreventing an adhesion of suspended cells on the sample carrier.

EXAMPLE 3

Examination of the Toxicity of Polyvinyl Alcohol with Respect to LivingMammalian Cells

Implementation:

The use of 0.5% (w/v) of PVA for the recovery of cells from a microlitersyringe was examined. Jurkat-cells were suspended in a concentration of5.69×10⁵ cells per ml in PBS with 0.5% PVA (w/v). 5 μl of thissuspension were drawn up into a 5 μL-syringe (Dynatech). 1 μl each weretransferred immediately and after 1 and 5 minutes, respectively, into awell of a Terasaki plate (NUNC, Wiesbaden). The cells were stained inthe plate with the live/dead stain kit (Molecular Probes, Leiden,Netherlands) and counted; thereby their vitality was simultaneouslyexamined. The recovery rate was determined.

Result:

More than 90% of the cells could be recovered—after 5 minutes aswell—from the syringe. They therefore had not adhered. The percentage ofdead cells initially, thus at 0 minutes, amounted to 0.8% and increasedafter 5 minutes only to 2.4%.

EXAMPLE 4

Examination of Cells in Microanalysis Systems Using a Cell Buffer withPolyvinyl Alcohol

Material:

The use of polyvinyl alcohol for the work with suspended cells inmicrochannels was examined with a Cytocon™ 300 device system. TheCytocon™ chips consist of glass and exhibit channels with a height of 40μm, a width of 200-600 μm, and a length of about 1-2 cm, wherebyspecific embodiments of the chips may also exhibit electrodes in thechannels. Cytocon™ sorter chips comprise, for example, electrodes asdielectric junctions in the microchannels for the sorting of thesuspended particles (in particular cells), in Cytocon™ porator chips theelectrodes are arranged in such a way, that cell fusion and/or cellporation can be carried out, and Cytocon™ loader chips, that exhibit twoelectrode levels in a closed microchannel, a dielectric field cage in achannel intersection, as well as electrodes developed as dielectricjunctions, are suitable for the performance of a stop-flow analysis aswell as the subsequent sorting of the analyzed particles.

Polyvinyl alcohol with a molecular weight of 30,000 g/mol to 70,000g/mol was used. The polyvinyl alcohol was dissolved in the buffer to0.5% (w/v) and was present during the entire manipulation of the cellsin the microchannels.

Implementation:

The cells were suspended in the buffer and injected into a Cytocon™chip. The suspension was transported within the channel system with acertain flow rate (“sample”) and at the chip exit accelerated with asheath fluid. The rinsed out cells were counted and the recovery ratecalculated from the initial concentration and the injected sampleamount.

Result:

Even though the cell amount used was only a few hundred cells and theflow rate in the microchannels was very slow (dwell time of the cells inthe microchannel about 2 minutes), the recovery rate of cells was veryhigh (table 1) and values around 100% were achievable.

EXAMPLE 5

Fractionation of Blood Cells (Separation of Lyphocytes from Red BloodCells)

Implementation:

A mixture of whole human blood, Jurkat T-lymphoma cells, and aPVA-containing buffer was prepared. The buffer contained 20% (v/v) PBS,80% (v/v) 0.3 M inositol solution, and 0.5% (w/v) PVA (30,000-70,000,Sigma). For a better visualization during the experiment, the Jurkatcells were labeled with 10 μM Calcein-AM™ (Molecular Probes).

The blood and the lymphoma cells were diluted as follows: The cellsamples were mixed to concentrations of 2.5·10⁶ Jurkat cells/ml and1.8·10⁷ red blood cells/ml. Samples of 0.3 μl were injected into aCytocon™ chip (microanalysis system).

The Cytocon™ chips used for this experiment consisted of glass andexhibited channels with a height of 40 μm, a width of 200-600 μm, and alength of about 1-2 cm. Additionally, the microanalysis system usedhere, comprised electrodes in one or several microchannels, that arearranged here as dielectric junction for the sorting of the cells.

The cells were separated based on differences in their size and theirdielectric properties via the so-called switch electrode (junction) ofthe Cytocon™ microanalysis system at a frequency of the electric fieldof 800 kHz and an amplitude of 3-6 V rms at a flow rate of 62-240 μm/s.Under these conditions, the Jurkat cells were deflected and collected infraction 1, while the red blood cells (RBC) could pass the junction andwere collected in fraction 2.

Jurkat cells and red blood cells (RBC) were counted in both fractions inorder to be able to determine the accumulation and the yield.

Result:

The accumulation factor for the Jurkat cells in fraction 1 is in anexemplary experiment 17.5. The yield of Jurkat cells in this fractionwas 93% in this case (table 2).

Overall (fractions 1 and 2), the cell recovery rate was almost 100%.

1. A method for inhibiting adhesion of particles to surfaces, saidmethod comprising: providing the particles comprising at least one ofeukaryotic cells, prokaryotic cells, and cellular components thereof;providing a substrate comprising a microanalysis system or syntheticmicroparticles, wherein the substrate has surfaces to which theparticles are capable of adhering; providing the particles in a solutioncomprising polyvinyl alcohol; and contacting the substrate with thesolution, wherein the polyvinyl alcohol is present as a solute in thesolution in an amount effective to inhibit adhesion of the particles tothe surfaces, wherein the concentration of the polyvinyl alcohol insolution is 0.01% (w/v) to 5% (w/v).
 2. A method for inhibiting adhesionof particles to surfaces, said method comprising: providing theparticles comprising at least one of viable eukaryotic cells and viableprokaryotic cells; providing a substrate comprising a microanalysissystem or synthetic microparticles, wherein the substrate has surfacesto which the particles are capable of adhering; providing the particlesin a solution comprising polyvinyl alcohol; and contacting the substratewith the solution, wherein the polyvinyl alcohol is present as a solutein the solution in an amount effective to inhibit adhesion of theparticles to the surfaces; wherein the concentration of the polyvinylalcohol in solution is 0.01% (w/v) to 5% (w/v); and wherein the cellsretain their viability.
 3. The method according to claim 1, wherein thesurfaces comprise channel surfaces of the microanalysis system.
 4. Themethod according to claim 1, wherein the surfaces comprise surfaces ofthe synthetic microparticles.